Throughout this application, various publications, patents, and published patent applications are referred to by an identifying citation; full citations for these documents may be found at the end of the specification immediately preceding the claims. The disclosures of the publications, patents, and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
Luminescence is the term commonly used to refer to the emission of light from a substance for any reason other than a rise in its temperature. In general, atoms or molecules emit photons of electromagnetic energy (e.g., light) when then move from an “excited state” to a lower energy state (usually the ground state); this process is often referred to as “radiative decay.” There are many causes of excitation. If exciting cause is a photon, the luminescence process is referred to as “photoluminescence.” If the exciting cause is an electron, the luminescence process is referred to as “electroluminescence.” More specifically, electroluminescence results from the direct injection and removal of electrons to form an electron-hole pair, and subsequent recombination of the electron-hole pair to emit a photon. Luminescence which results from a chemical reaction is usually referred to as “chemiluminescence.” Luminescence produced by a living organism is usually referred to as “bioluminescence.” If photoluminescence is the result of a spin-allowed transition (e.g., a single-singlet transition, triplet-triplet transition), the photoluminescence process is usually referred to as “fluorescence.” Typically, fluorescence emissions do not persist after the exciting cause is removed as a result of short-lived excited states which may rapidly relax through such spin-allowed transitions. If photoluminescence is the result of a spin-forbidden transition (e.g., a triplet-singlet transition), the photoluminescence process is usually referred to as “phosphorescence.” Typically, phosphorescence emissions persist long after the exciting cause is removed as a result of long-lived excited states which may relax only through such spin-forbidden transitions.
Electrochemiluminescence (“ECL”), also referred to as electrogenerated chemiluminescence, generally pertains to the emission of photons of electromagnetic radiation (e.g., light) from an electronically excited chemical species which has been generated electrochemically. In a simple example, species A, in the ground state, is first electrochemically reduced to form a reduced species A− that may then diffuse from the electrode surface. Similarly, a species A is electrochemically oxidized to form an oxidized species A+. The reduced species A− and the oxidized species A+ then diffuse together and react to form an electronically excited species, A*, and a ground state species, A. The electronically excited species, A*, then relaxes to the ground state by emitting a photon.
A+e−→A−
A−e−→A+
A−+A+→A*+A
A*→A+hv 
In common similar examples, a coreactant, CR, reacts with either an electrochemically generated reduced or oxidizes species, A+ or A−, to form an electronically excited species A*, when then relaxes to the ground state by emitting a photon.
A + e− → A−A − e− → A+A− + CR → A* + CR′A+ + CR → A* + CR′A* → A + hvA* → A + hv
ECL was first observed in the late 1920's and was investigated in detail during the late 1960's and 1970's. A number of literature reviews pertaining to the nature ECL (e.g., the emitting state, the emission mechanism, the emission efficiency) have been published. See, for example, Knight et al., 1994 and references cited therein.
ECL of polyaromatic hydrocarbons in both aqueous and non-aqueous media has been widely studied. Examples of such compounds include naphthalene, anthracene, phenanthrene, pyrene, chrysene, perylene, coronene, and rubrene.
A typical example is the ECL of 9,10-diphenylanthracene (“DPA”). A double potential step is applied to a platinum electrode, producing anodic oxidation products (i.e., the radical cation, DPA*+) at the positive potential and cathodic reduction products (i.e., the radical anion, DPA*−) at the negative potential. The products undergo electron transfer to yield DPA and electronically excited (singlet) state 1DPA*, which emits a photon via chemiluminescence (in this case, fluorescence). In this example, the emitting state is formed directly upon electron transfer (so called “S-route”).
DPA−e−→DPA*+ (electro-oxidation)
DPA+e−→DPA*− (electro-reduction)
DPA*++DPA*−→DPA+1DPA* (electron transfer)
1DPA* →DPA*−+hv (chemiluminescence)
ECL may also be generated using polyaromatic hydrocarbons in combination with other chemical species which may act as suitable donor or acceptor molecules in the electron transfer step. For example, another common ECL system involves DPA and the donor species, N,N′,N″, N′″-tetramethyl-para-phenylenediamine (“TMPD”). In this case, the emitting state is formed in a second (inefficient) triplet-triplet annihilation step from the product of the first electron transfer step (so-called “T-route”).
TMPD−e−→TMPD*+ (electro-oxidation)
DPA+e−→DPA*− (electro-reduction)
TMPD*++DPA*−→TMPD+3DPA* (electron transfer 1)
3DPA*+3DPA*→DPA+1DPA* (electron transfer2)
1DPA*→DPA*−+hv (chemiluminescence)
ECL of inorganic and/or organometallic compounds has also been widely studied. An important class of such compounds are the 2,2′-bipyridine (“bpy”) complexes of ruthenium and osmium, such as Ru(bpy)32+ and Os(bpy)32+. Other examples of such compounds include tricarbonyl(chloro)(1,10-phenanthroline) rhenium(I), square planer platinum(II) complexes, Cr(bpy)32+, multinuclear complexes such as Pt2(diphosphonate)44−, and clusters such as Mo6Cl122−. See, for example, Knight et al., 1994.
Most investigations of inorganic and/or organometallic compounds have centered on Ru(bpy)32+ and related compounds primarily due to their intrinsic, and somewhat exceptional, properties, including the ability to emit luminescence at room temperature in aqueous solution, the ability to undergo reversible one-electron transfer reactions at easily attainable potentials, leading to sufficiently stable reduced or oxidized species, insensitivity to the presence of oxygen, and an annihilation efficiency of nearly 100% under certain conditions. For example, if a solution of Ru(bpy)32+ is subjected to a cyclic double-step potential alternating between the oxidation and reduction potential of the complex, an orange emission is observed (at ˜620 nm).
Ru(bpy)32+−e−→Ru(bpy)33+ (electro-oxidation)
Ru(bpy)32++e−→Ru(bpy)3+ (electro-reduction)
Ru(bpy)3++Ru(bpy)33+→Ru(bpy)32++Ru(bpy)32+* (electron transfer)
Ru(bpy)32+*→Ru(bpy)32++hv (chemiluminescence)
ECL may also be generated using Ru(bpy)32+ in combination with strong oxidizing or reducing species in solution; in this way, only half of the double-step oxidation-reduction cycle need be applied. For example, coreactants peroxodisulfate (i e., S2O82−,persulfate) and oxalate (i.e., C2O42−) are irreversibly reduced or oxidized, respectively, to form oxidizing SO4*− or reducing CO2*−ions. For example,
Ru(bpy)32++e−→Ru(bpy)3+ (electro-reduction)
S2O82−+e−→SO42−+SO4*− (electro-reduction)
SO4*−+Ru(bpy)3+→SO42−+Ru(bpy)32+* (electron transfer)
SO4*−+Ru(bpy)32+→SO42−+Ru(bpy)33+ (electron transfer)
Ru(bpy)3++Ru(bpy)33+→Ru(bpy)32++Ru(bpy)32+* (electron transfer)
Ru(bpy)32+*→Ru(bpy)32++hv (chemiluminescence)
In a similar manner, ECL may also be generated using Ru(bpy)32+ in combination with coreactants such as amines, or compounds containing amine groups, which act as reducing agents. In general, emission from the Ru(bpy)32+ ECL reaction with amines increases in the order, primary <secondary<tertiary. Aliphatic or alicylic amines are generally more efficient than aromatic amines. An example of a commonly used amine is tri-n-propylamine (i.e., N(CH2CH2CH3)3, “TPAH”). See, for example, Leland et al., 1990. It is commonly believed that a proton is lost from an α-carbon of one propyl group upon electro-oxidation and subsequent reaction, to yield TPA* (i.e., (CH3CH2CH2)2N(CHCH2CH3)* ). The ECL sequence is summarized by the reactions below.
Ru(bpy)32+−e−→Ru(bpy)33+ (electro-oxidation)
TPAH−e−→[TPAH]+→TPA*+H+ (electro-oxidation and reaction)
Ru(bpy)33++TPA* →Ru(bpy)32+*+products (electron transfer)
Ru(bpy)32+*→Ru(bpy)32++hv (chemiluminescence)
In this way, ECL of Ru(bpy)32+ has been employed in the determination of a wide range of coreactants. For example, Ru(bpy)32+ ECL has been effectively used to determine oxalate and persulfate to levels as low as 10−13 moles/liter. Similarly, Ru(bpy)32+ ECL has been employed in the determination of aliphatic amines, alicylic amines (such as sparteine, nicotine, and atropine), drugs such as erythromycin (which has a trialkylamine group), amino acids (such as valine and proline), and proteins. This in turn has led to the implementation of Ru(bpy)32+, as well as other ruthenium and osmium chelates, as sensitive ECL labels for chemical and biochemical assays.
Chemical and biological assays generally involve contacting the analyte of interest with a pre-determined non-limiting amount of one or more assay reagents, measuring one or more properties of a resulting product (the detection product(s)), and correlating the measured value with the amount of analyte present in the original sample, typically by using a relationship determined from standard samples containing known amounts of analyte of interest in the range expected for the sample to be tested. Typically, the detection product incorporates one or more detectable labels, which are provided by one or more assay reagents. Examples of commonly used labels include radioactive isotope labels, such as 125I and 32P; enzyme (e.g., peroxidase, β-galactosidase) and enzyme substrate labels; fluorescent labels (e.g., fluorosceines, rhodamines); electron-spin resonance labels (e.g., nitroxide free radicals); immunoreactive labels (e.g., antibodies, antigens); and labels which are one member of a binding pair (e.g., biotin-avidin, biotin-streptavidin). Sandwich assays typically involve forming a complex in which the analyte of interest is sandwiched between one assay reagent which is ultimately used for separation (e.g., antibody, antigen, one member of a binding pair) and a second assay reagent which provides a detectable label. Competition assays typically involve a system in which both the analyte of interest and an analog of the analyte compete for a binding site on another reagent (e.g., an antibody), wherein one of the analyte, analog, or binding reagent possess a detectable label.
Recently, ECL labels have become more common in chemical and biological assays. For example, ECL labels (e.g., those containing a Ru(bpy)32+ moiety) can be modified by attaching reactive groups (e.g., to one or more of the bipyridyl ligands) to form activated labeling reagents for proteins, nucleic acids, and other molecules. This approach offers many advantages over other detection systems, such as 32P radiolabeling, including, but not limited to, many of the following: (1) the absence of radioactive isotopes thereby reducing the problems associated with sample handling and disposal; (2) very low detection limits for the ECL label, often as low as 0.2 picomolar (2×10−13 M), since each label can emit several photons per measurement cycle; (3) a dynamic range for label quantification which often extends over six orders of magnitude; (4) extremely stable labels often with long shelf lives; (5) low molecular weight labels (˜1000 atomic units) which may be coupled to proteins, oligonucleotides, etc., often without affecting immunoreactivity, solubility, ability to hybridize, etc.; (6) high selectivity and low background, since the ECL reaction sequence is initiated electrochemically and only those species with appropriate electrochemical properties in the proximity of the electrode are detected; and (7) simple and rapid measurement, typically requiring only a few seconds.
In recent years, ECL has been exploited in the development of immunoassays and DNA probe analysis. See, for example, Blackburn et al., 1991, Kenten et al., 1991, 1992, Leland et al., 1992, and Yost, 1993.
In a typical and well known DNA separation assay employing ECL, the target oligonucleotide is amplified (e.g., using PCR) using a biotin-containing primer oligonucleotide to yield an increased concentration of target oligonucleotides which comprise a biotin moiety; an excess of an oligonucleotide hybridization probe to which is attached an ECL label, and which hybridizes to the target oligonucleotides to form hybridized probe-target duplexes, is then added; streptavidin coated beads, which strongly and selectively bind the biotin-containing duplexes, are added; the beads are separated from the mixture (e.g., magnetically, gravimetrically), thereby removing the excess labeled oligonucleotide hybridization probe; and the target oligonucleotide, which is bound to the beads and which is hybridized to an ECL-labeled hybridization probe, is detected and/or quantified using ECL.
Blackburn et al. (1991) apparently disclose the use of an N-succinimidyl ester derivative of Ru(bpy)32+ as a means for attaching an ECL label to an oligonucleotide hybridization probe. By using a biotin-labeled oligonucleotide primer, the polymerase chain reaction (“PCR”) amplification products could be separated by binding to streptavidin coated magnetic beads. Once separated, the Ru(bpy)32+ labeled oligonucleotide probe was hybridized to the bound PCR products, and detected by ECL.
Kenten et al. (1991) apparently similarly disclose the use of ECL assays of PCR amplified products from oncogenes, viruses, and cloned genes. In one assay, a Ru(bpy)32+ label was attached to one or both of the oligonucleotide primers; following amplification, binding, and separation, the PCR products were detected by ECL. In another assay, an oligonucleotide probe having an attached Ru(bpy)32+ label was hybridized to magnetic bead-bound PCR products, the excess probe was removed by washing, and the hybridized product detected by ECL. In a third assay, an oligonucleotide probe having an attached Ru(bpy)32+ label was hybridized to unbound PCR products, the hybridized product was bound to magnetic beads, the excess probe was removed by washing, and the hybridized product detected by ECL. In each case, the desired product was detected by the presence of an ECL label.
Kenten et al. (1992) apparently disclose “binding assays” which employ ECL labels. Apparently, a complex comprising the analyte of interest, an ECL label, and particle is formed, and the presence of this complex is subsequently detected by ECL.
Chemical and biological assays may often be conveniently classified as “separation assays” or “non-separation assays.” Generally, in separation assays, the detection product(s) is physically separated from other products and/or unreacted analyte of interest and unreacted assay reagents. (For example, it is often necessary to physically separate the detection product so that only those labels which are part of the detection product are detected, and not those of the excess labeling reagent.) The amount of analyte may then be determined either directly from the amount of labeled detection product, or indirectly from the amount of unused labeling reagent. Separation may often be achieved by exploiting a selective binding reaction between members of a binding pair (e.g., biotin-avidin, antibody-antigen, oligonucleotide hybridization probe-oligonucleotide). For example, a labeled detection product having one member of a binding pair may be first formed in a fluid phase (e.g., in solution), and separation may then be effected, for example, by capture of the ECL labeled detection product by a solid phase reagent having the other member of the binding pair; the detection product may then be recovered by washing the solid phase free of unreacted analyte and reagents. Many other separation strategies employing binding pairs are well known in the art.
Assays which do not require a separation step are highly desirable, as they typically require less sample manipulation and are often readily adapted to “real time” assays. Such assays may often be conveniently classified as “non-separation assays.” In non-separation assays, the detection product is typically not physically separated from unused assay reagents and unused analyte. Instead, the presence of the detection product is typically detected by a property which at least one of the assay reactants acquires or loses only as a result of contacting the analyte of interest. A number of such non-separation assays have been developed.
In one example of a non-separation assay, both an enzyme and an enzyme inhibitor is used. Upon contacting the analyte of interest, the enzyme and enzyme inhibitor are either brought together (to reduce enzyme activity) or separated (to increase enzyme activity). Any change in enzyme activity is then correlated with the presence and/or amount of the analyte of interest. See, for example, Yoshida et al., 1980, and Zuk et al., 1980.
In another example of a non-separation assay, both a chromophore and a chromophore modifier is used. Again, upon contacting the analyte of interest, the chromophore and chromophore modifier are either brought together or separated, thereby yielding a change in color or a change in intensity of a specified color. Any change in color and/or intensity of color is then correlated with the presence and/or amount of the analyte of interest. See, for example, Zuk et al., 1980.
In yet another example of a non-separation assay, both a fluorophore and a fluorophore quencher is used. Upon contacting the analyte of interest, the fluorophore and fluorophore quencher are either brought together (to reduce fluorescence) or separated (to increase fluorescence). See, for example, Ullman et al., 1976; Ullman, 1979; Zuk et al., 1981; and Ullman et al., 1981. More recent examples of photoluminescence assays (e.g., fluorescence assays) which exploit photoluminescence quenchers are discussed below.
Tyagi et al. (1996) apparently disclose an assay for oligonucleotides which employs a particular oligonucleotide probe (referred to as a “molecular beacon”) which possesses both a fluorophore (i.e., a label) and a fluorescence quencher. In the absence of the target oligonucleotide, portions of the oligonucleotide probe hybridize with itself, bringing the fluorophore and the fluorescence quencher into close proximity; in this form, no fluorescence signal is observed. In the presence of the target oligonucleotide, the oligonucleotide probe de-hybridizes and preferentially hybridizes with the target oligonucleotide, and in doing so, separates the fluorophore and the fluorescence quencher; in this form, a fluorescence signal is observed. Thus, fluorescence is only observed from those oligonucleotide probes which are hybridized with the target oligonucleotide. In this way, it is not necessary to remove the unhybridized oligonucleotide probes prior to measuring the fluorescence signal.
Heid et al. (1996) apparently disclose a real-time quantitative assay for DNA analysis using dual-labeled fluorogenic hybridization probes. An oligonucleotide probe is prepared having a first fluorescent dye (FAM, 6-carboxyfluorescein) which acts as a reporter, and a second fluorescent dye (TAMRA, 6-carboxy-tetramethylrhodamine) which quenches the emission spectra of the first fluorescent dye. The 5′-specific exo-nuclease activity of the Taq polymerase causes only those probes which have hybridized with a target oligonucleotide to be degraded, releasing the two dyes, and resulting in an increase in the FAM fluorescent emission.
Wittwer et al. (1997) apparently disclose methods for continuous fluorescence monitoring of PCR products during amplification. In one assay, commercially available dual labeled oligonucleotide probes possessing both a “donor” moiety (e.g., fluorescein) and an “acceptor” moiety (e.g., rhodamine) are hybridized to a target oligonucleotide. The close proximity of the acceptor apparently attenuates the fluorescence signal from the donor. A polymerase having 5′-specific exo-nuclease activity is added, and, during polymerization, the oligonucleotide probe is degraded, releasing both the donor and the acceptor. No longer in close proximity to the acceptor, the donor then yields an increased fluorescence signal. In another assay, based on resonance energy transfer, two different oligonucleotide probes were prepared, one having a “donor” moiety (e.g., fluorescein) and one having an “acceptor” moiety (e.g., the cyanine dye Cy5®). The two oligonucleotide probes were selected so that, when hybridized to the target oligonucleotide, the donor and acceptor moieties are brought into close proximity. When the donor is photoexcited, some or all of its energy is transferred to the acceptor, and the fluorescence signal from the acceptor increases. (See also, for example, Maliwal, et al., 1995). In this way, the target oligonucleotide is detected and quantified by an increase in the fluorescence signal from the acceptor.
Unlike the quenching of ECL, the quenching of photoluminescence has been widely studied, and many compounds are known to quench photoluminescence under a variety of conditions. In sharp contrast, only a few compounds are known to efficiently quench ECL, and many of those which are well known (e.g., methylviologen carboxylate) either only poorly quench ECL or are impractical for use in assays.
The inventors have discovered that certain other classes of compounds strongly quench ECL, such as compounds comprising at least one benzene moiety, and, more particularly, compounds comprising at least one phenol moiety, quinone moiety, benzene carboxylic acid, and/or benzene carboxylate moiety.
The ECL properties of such compounds have not been widely studied. The use of strongly fluorescent compounds, such as anthracenes, to increase ECL emission is known, and indeed widely used in the common ECL assays. Chmura et al. (1994) apparently examined assays for antioxidants and free radical scavengers, such as citrate, which relied on these compounds' ability to quench of anthracene-sensitized ECL. Kricka et al. (1991) apparently describe the use of p-iodophenol as a chemiluminescence enhancer, rather than a quencher. Hill et al. (1988) apparently examined the ECL emission from a number of dansylated derivatives in an effort to adapt ECL to reverse-phase liquid chromatography (RPLC). They apparently examined the effect of the dansyl group (i.e., 5-dimethylamino-1-naphthalanesulfonyl), which is a well known fluorescent label, on the ECL emission of a number of amino acids and phenolic compounds, and found that the presence of a dansyl group increased the ECL emission of many of the compounds tested. In their study of the ECL of osmium complexes, Abruna et al. (1985) apparently describe the quenching of ECL of an Os(bpy)2diphos+2 species by a ferricenium species (the oxidized form of a ferrocene), a species comprising two cyclopentadienyl ions (i.e., C5H5−) and a sandwiched ferrous (i.e., Fe+2) ion.
Using the efficient ECL quenchers disclosed herein, assays may be developed which employ an ECL label and an ECL quencher and which permit, inter alia, assays such as non-separation assays which offer many, if not all, of the advantages offered by ECL detection methods over other detection methods. Thus, the present invention broadly pertains to certain classes of chemical moieties which strongly quench ECL, and the use of these ECL quenchers, for example, in ECL assays which employ an ECL label and an ECL quencher. One class of such quenching moieties are those which comprise at least one benzene moiety. Sub-classes of such quenching moieties are those which comprise at least one phenol moiety, quinone moiety, benzene carboxylic acid, and/or benzene carboxylate moiety.